CN115551515A

CN115551515A - Forsythiaside targeting 3CLpro, derivative thereof and anti-neocorolla application

Info

Publication number: CN115551515A
Application number: CN202180005782.6A
Authority: CN
Inventors: 杨子峰; 富力; 蒋革; 钟南山; 杨威; 马钦海; 惠敏; 李楚芳; 王硕; 潘蔚倚; 鲁岐; 宋瑷瑷; 侯集瑞; 李润峰; 鲁明明; 王英平; 柳洋; 刘国友; 付文斐; 冯雪
Original assignee: Dalian Fu Sheng Natural Pharmaceutical Development Co ltd
Current assignee: Dalian Fu Sheng Natural Pharmaceutical Development Co ltd
Priority date: 2020-03-09
Filing date: 2021-02-09
Publication date: 2022-12-30
Anticipated expiration: 2041-02-09
Also published as: US20230101803A1; EP4119146A4; EP4119146A1; JP2023510015A; JP7430803B2; BR112022015046A2; CN115551515B; WO2021179878A1; CN113368121A; CA3168457A1; ZA202211004B

Abstract

Application of phillyrin and derivatives thereof targeting 3CLpro protein inhibiting COVID-19 virus, and phillyrin and phillygenin composition in preparation of medicine for resisting coronavirus or treating diseases caused by coronavirus. Wherein the coronavirus is a COVID-19 virus, and the disease caused by the coronavirus is COVID-19.

Description

Forsythiaside targeting 3CLpro, derivative thereof and anti-neocorolla application

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to phillyrin and/or a derivative thereof of 3CLpro protein for targeted inhibition of COVID-19 virus, application of phillyrin and phillygenin composition in preparation of anti-coronavirus (especially COVID-19 virus) medicines, and the like.

Technical Field

The disease causing source of COVID-19, namely 2019 coronavirus (also called novel coronavirus pneumonia), is COVID-19 virus (also called novel coronavirus), which belongs to coronavirus, and has higher homology with SARS outbreak in 2003. At present, no specific medicine for treating COVID-19 exists. In fact, no specific drug has been available to date for SARS that has already developed.

The present inventors have conducted intensive studies on extracts of Chinese herbs and have discovered, occasionally and surprisingly, that a pharmaceutical composition comprising forsythin and less forsythiaside has a synergistic, especially synergistic, pharmaceutical effect against viruses. For example, chinese patent application CN105362283A discloses a forsythin/forsythiaside composition and its use in alleviating or/and treating viral diseases, wherein the viral diseases are viral diseases caused by influenza virus, parainfluenza virus, coxsackievirus CoxA16, respiratory syncytial virus RSV, herpes simplex virus HSV-I, herpes simplex virus HSV-II, herpes simplex virus CVB3, adenovirus ADV or enterovirus EV 71.

However, there are many drugs with antiviral action, and the action mechanism is diversified, so that it is impossible to predict which of the existing antiviral drugs have inhibitory effect on the newly emerging COVID-19 virus. In particular, because the coronavirus is quite different from the above-mentioned phillyrin/phillygenin composition, and there is no current discussion about the COVID-19 virus and the therapeutic mechanism thereof, the skilled person cannot predict that the phillyrin/phillygenin composition has an inhibitory effect on the coronavirus (especially the COVID-19 virus).

In addition, the effect of forsythin, forsythiaside or derivatives thereof on the hydrolase (3-chylotrysin-like cysteine protease, abbreviated as 3CL pro protein) of the COVID-19 virus has not been reported.

However, the inventor surprisingly found that the phillyrin/phillygenin composition has a good inhibitory effect on the COVID-19 virus, and combined with the high safety, the clinical approval of other indications is approaching completion, so that the phillyrin/phillygenin composition has a prospect of becoming a medicament for treating COVID-19 and can be rapidly put into clinical use; furthermore, the inventor finds that the phillyrin, the phillygenin or the derivatives of the phillyrin and the phillygenin have a targeted inhibition effect on the 3CL pro protein of the COVID-19 virus, and can be used as an inhibitor of the protein.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a novel reagent for targeting 3CLpro protein inhibiting COVID-19 virus and provide a medicament for resisting coronavirus (especially COVID-19 virus) or treating diseases caused by coronavirus (especially COVID-19).

In particular, in a first aspect, the invention provides the use of phillyrin and/or a derivative thereof in the preparation of an agent for inhibiting the 3CLpro protein of the COVID-19 virus. The agent for inhibiting 3CLpro protein of COVID-19 virus comprises phillyrin and/or its derivative.

The use of the first aspect of the invention may be the sole use of forsythin or a derivative thereof or the combined use of forsythin and a derivative thereof, for example the use of a forsythin/forsythiaside composition. In the use of the first aspect of the invention, the inhibition may be in vivo inhibition or in vitro inhibition.

Preferably in the use of the first aspect of the invention, the derivative is KD-2-GLU or KD-2-SO ₃ H。

Accordingly, in a second aspect, the present invention also provides a method of inhibiting the 3CLpro protein of covd-19 virus, comprising the step of contacting the 3CLpro protein of covd-19 virus with phillyrin and/or a derivative thereof. The method may be an in vivo method or an in vitro method.

Preferably in the method of the second aspect of the invention, the derivative is KD-2-GLU or KD-2-SO ₃ H。

In a third aspect, the invention provides the use of a forsythin/forsythiaside composition in the manufacture of a medicament for the treatment of an anti-coronavirus disease, and further provides the use of a forsythin/forsythiaside composition in the manufacture of a medicament for the treatment of a disease caused by a coronavirus disease. The anti-coronavirus may be an anti-coronavirus in vitro, e.g. to inhibit the proliferation of a coronavirus in vitro, but is preferably an anti-coronavirus in vivo, the latter being for the treatment of a disease caused by a coronavirus.

In this context, unless indicated to the contrary, "forsythin/forsythin", "forsythin and forsythin" and "forsythin and forsythin composition" are used interchangeably and refer to a composition consisting of forsythin and forsythin, i.e. forsythin/forsythin as a whole. Preferably, in the invention, the weight ratio of phillyrin to phillyrin is 2-98: 2 to 98, preferably 80 to 98:2 to 20, more preferably 90 to 98:2 to 10, such as 90.

Preferably in the use of the third aspect of the invention, the coronavirus is a COVID-19 virus, or the coronavirus-induced disease is COVID-19. Preferably, the forsythin/forsythiaside composition inhibits the 3CLpro protein of the COVID-19 virus, i.e. in the use of the third aspect of the invention, the forsythiaside/forsythiaside composition is directed against the COVID-19 virus or to the treatment of diseases caused by the COVID-19 virus by inhibiting the 3CLpro protein of the COVID-19 virus.

The phillyrin/phillygenin composition can be used in combination with other anti-coronavirus (e.g., COVID-19 virus) or therapeutic agents for coronavirus-induced diseases (e.g., COVID-19) or alone. The inventor researches to find that the mechanism of the phillyrin/phillygenin composition for resisting the COVID-19 virus comprises inhibiting the 3CLpro protein of the COVID-19 virus.

Preferably the use of the third aspect of the invention is the use of a forsythin/forsythiaside composition as the sole pharmaceutically active ingredient, i.e. the sole active ingredient in the medicament is a forsythin/forsythiaside composition. That is, the third aspect of the present invention preferably provides use of the forsythin/forsythiaside composition as the sole pharmaceutically active ingredient in the manufacture of a medicament for use against coronavirus (e.g. COVID-19 virus), or use of the forsythin/forsythiaside composition as the sole pharmaceutically active ingredient in the manufacture of a medicament for use in the treatment of a disease caused by coronavirus (e.g. COVID-19).

The medicament may be formulated into a pharmaceutical preparation including a pharmaceutically acceptable carrier. As is well known to those skilled in the art. Preferably in the use of the third aspect of the invention, the medicament is in the form of a tablet, capsule, pill, powder, granule, syrup, solution, emulsion, injection, spray, aerosol, gel, cream, cataplasm, rubber patch or plaster.

Accordingly, the present invention also provides a second pharmaceutical formulation of the forsythin/forsythiaside composition and a method of treatment using the same. In a fourth aspect, the present invention provides a phillyrin/phillygenin composition for use against a coronavirus (e.g., COVID-19 virus). The present invention also provides forsythin/forsythiaside compositions for the treatment of coronavirus-induced diseases (e.g., COVID-19); alternatively, the invention also provides a phillyrin/phillygenin composition for inhibiting the 3CLpro protein of the COVID-19 virus.

Further, the present invention provides medicaments comprising the phillyrin/phillygenin composition for use against coronaviruses (e.g., COVID-19 virus). The invention also provides medicaments comprising the phillyrin/phillygenin composition for use in treating a coronavirus-induced disease (e.g., COVID-19); alternatively, the invention also provides a medicament comprising the phillyrin/phillygenin composition, which is used for inhibiting the 3CLpro protein of the COVID-19 virus.

Preferably, in the medicament comprising the forsythin/forsythiaside composition, forsythin/forsythiaside is the only pharmaceutically active ingredient in the medicament.

Also preferably, the medicament comprising the forsythin/forsythiaside composition is in the form of a tablet, capsule, pill, powder, granule, syrup, solution, emulsion, injection, spray, aerosol, gel, cream, cataplasm, rubber patch or patch.

In a fifth aspect, the present invention provides a method for combating coronavirus (e.g., COVID-19 virus), which comprises administering to a patient in need thereof an effective amount of a forsythin/forsythin composition, and accordingly, the present invention also provides a method for the manufacture of a medicament for the treatment of a disease caused by coronavirus (e.g., COVID-19), which comprises administering to a patient in need thereof an effective amount of a forsythin/forsythin composition. Preferably, the phillyrin/phillygenin composition inhibits the 3CLpro protein of the COVID-19 virus to combat the COVID-19 virus or treat diseases caused by the COVID-19 virus.

Further, the present invention provides a method for resisting the COVID-19 virus, which comprises administering an effective amount of a medicament comprising a forsythin/forsythiaside composition to a patient in need thereof, and accordingly, the present invention also provides a method for treating a disease caused by coronavirus (e.g., COVID-19) comprising administering an effective amount of a medicament comprising a forsythin/forsythiaside composition to a patient in need thereof.

In this context, the dose (effective amount) and form to be administered are generally determined by a physician according to the particular circumstances of the patient, such as age, body weight, sex, duration of illness, physical condition, severity of infection, and the like. As the patient's condition may vary, and hence the dosage administered, the appropriate amount is within the capabilities of the clinician. The administration form is determined according to the dosage form of the pharmaceutical composition, and suitable administration forms include oral, parenteral injection, mucosal, intramuscular, intravenous, subcutaneous, intraocular, intradermal, or transdermal administration forms, and preferably oral administration forms.

Preferably, in the method, the forsythin/forsythiaside composition is the only pharmaceutically active ingredient in the medicament.

Also preferably, in the method, the medicament is in the form of a tablet, capsule, pill, powder, granule, syrup, solution, emulsion, injection, spray, aerosol, gel, cream, cataplasm, rubber patch or patch.

The invention has the advantages that the invention provides the reagent and the method for inhibiting the 3CLpro protein of the COVID-19 virus; the phillyrin/phillygenin composition can effectively inhibit coronavirus such as COVID-19 virus and the like at low concentration, wherein the combination of the phillyrin and the phillygenin has a synergistic effect, has a prospect of being a medicine for treating coronavirus such as COVID-19 and can be rapidly put into clinical use.

For the purpose of facilitating understanding, the present invention will be described in detail below with reference to specific embodiments and the accompanying drawings. It is to be expressly understood that such description is intended as an illustration and not as a definition of the limits of the invention. Many variations and modifications of the invention will be apparent to those skilled in the art in light of the teachings of this specification. In addition, the present invention incorporates publications which are intended to more clearly describe the invention, and which are incorporated herein by reference in their entirety as if reproduced in their entirety.

Drawings

FIG. 1 shows the structures of phillyrin and its derivatives.

FIG. 2 is a schematic representation of the binding of KD-1 to the 3CLpro target protein of COVID-19.

FIG. 3 is a mimetic of the binding of KD-2-GLU to the COVID-19 target protein.

FIG. 4 shows KD-2-SO ₃ H and COVID-19 3CLpro target protein binding simulation diagram.

FIG. 5 shows the results of cloning, expression and purification of the 3CL pro protein.

FIG. 6 is a UV analysis of 3CLpro protein with KD-1.

FIG. 7 is a UV analysis chart of 3CLpro protein and KD 2-GLU.

FIG. 8 shows the interaction of 3CLpro protein with KD2-SO ₃ Ultraviolet analysis of H.

Detailed Description

The invention is further illustrated by the following examples. Unless otherwise specified, the methods used in the examples are described in the technical literature of the art and in the code documents of the drug administration, and the instruments, raw materials and reagents are commercially available.

EXAMPLE 1 Studies of phillyrin/phillygenin compositions against COVID-19 Virus in vitro

1 test drug: a forsythin/forsythiaside composition, wherein the weight ratio of forsythin to forsythiaside is 90.

2, cell: veroE6 cells (national focus laboratory of respiratory diseases, guangzhou respiratory health institute).

3, virus: the COVID-19 virus SARS-CoV-2 strain (BSL-3 laboratory (advanced disease and pathogenic microorganism research laboratory of respiratory disease national center for Key laboratory)) has a titer of TCID50=10 ^-6 /100μL。

4 experimental procedures (the experimental procedures were all completed in the BSL-3 laboratory):

(1) Sterile 96-well culture plate, 100. Mu.L of 2X 10 concentration per well ⁵ cells/mL VeroE6 cells, 37 ℃ 5% CO ₂ Culturing for 24 hours;

(2) Adding 100. Mu.L/well of 100TCID50 virus solution to the culture plate experimental group and virus control group, and 5% CO at 37% ₂ Adsorbing for 2 hours by an incubator;

(3) After 2h, discarding the cell culture solution in the 96-well culture plate; diluting phillyrin/phillygenin composition into a series of concentrations shown in Table 1, each concentration is 3 multiple wells, and adding the above medicinal liquid into 100 μ l/well;

(4) Simultaneously setting a cell control, a blank control (solvent control) and a virus control (negative control);

(5) Cell 37 ℃,5% ₂ Incubating in an incubator for 3-4 days;

(6) Cytopathic effect (CPE) was observed under an optical microscope and the degree of cytopathic effect was recorded according to the following 6-point scale: "-" no lesions appeared; "±" means less than 10% cytopathic effect; "+" is about 25% of the cellular pathology; "+ +" indicates about 50% of cellular pathology; "+++" indicates that about 75% of the cells are diseased: "+ ++" indicates that 75% or more of the lesions are diseased. The half maximal effective concentration (IC 50) was calculated using the Reed-Muench method or GraphPad prism 5.0.

5, experimental results:

the experimental results are shown in Table 1-1, the phillyrin/phillygenin composition can effectively inhibit the COVID-19 virus at low concentration, and the half inhibitory concentration IC is calculated ₅₀ 63.90. Mu.g/mL (while half the inhibitory concentration IC of the phillyrin pure product studied at the same time) ₅₀ 179.1 mu g/mL, but the inhibitory effect of the pure forsythiaside is worse), the combination of the two components of the forsythiaside and the forsythiaside in the composition has a synergistic effect, so the composition is expected to be used for treating COVID-19.

TABLE 1-1

EXAMPLE 2 Studies of phillyrin/phillygenin compositions against HCoV-229E in vitro

The coronavirus HCoV-229E is less pathogenic and typically causes only respiratory symptoms similar to the common cold. This example was carried out essentially as described in example 1, except that the coronavirus used was HCoV-229E (Guangzhou customs technology center BSL-3 laboratory (national institute of respiratory diseases, high therapeutic pathogenic microorganisms laboratory) with a titer of TCID50=10 ^-5.5 100 μ L, titer of 100TCID50 using virus; the phillyrin/phillygenin compositions were diluted to a range of concentrations as shown in table 2-1. The experimental operation is completed in a BSL-3 laboratory.

The experimental results are shown in Table 2-1, and the phillyrin/phillygenin composition can effectively inhibit HCoV-229E at low concentration, and the half inhibitory concentration IC is calculated ₅₀ 64.53. Mu.g/mL (while the phillyrin studied was pure)The product and the pure product of the forsythiaside have poorer inhibitory effect), the combination of the two components of the forsythiaside and the forsythiaside in the composition has the synergistic effect, and therefore, the composition is expected to be used for treating diseases caused by HCoV-229E.

TABLE 2-1

EXAMPLE 3 study of molecular mechanisms of forsythin and its derivative COVID-19 Virus

1. Background of the experiment

The Spike protein (S protein) on the surface of the COVID-19 virus and the hydrolase (3-chortypin-like cysteine protease, 3CL protein for short) of the COVID-19 virus are very important links in the life cycle process, the COVID-19 life cycle utilizes the combination of the S protein on the surface of the COVID-19 and angiotensin converting enzyme 2 (ACE 2), the S protein invades cells to release genetic material RNA through the endocytosis process of the cells, and the genetic material RNA is successfully replicated to generate the virus under the participation of the 3CL protein.

The virtual screening mainly applies a molecular docking technology, and is to simulate the process of drug screening on a computer, acquire the molecular structure of a drug action target when the screening is implemented, calculate the binding capacity of small molecules in a compound library and the target by a molecular simulation means, and predict the physiological activity of a candidate compound.

The experiment utilizes the Glide molecular docking technology to virtually screen the optimal binding effect of phillyrin (KD-1) and derivatives thereof on 3CL pro target protein, researches the binding of KD-1 and derivatives thereof on the target protein in vitro based on the screening result, and clarifies the intermolecular interaction and binding mode and the structure-activity relationship of KD-1 and derivatives thereof; through the activity measurement of KD-1 and the derivatives thereof on target protein, the molecular mechanism of KD-1 and the derivatives thereof as potential inhibitors of COVID-19 is revealed.

2. Experimental methods

Molecular docking of 1KD-1 and derivatives thereof to target proteins

(1) System preparation: the SARS-CoV-2 major protease (3 CL pro protein) was obtained from the protein database (PDB ID:6LU 7)

A crystal structure. The structure of the enzyme was pre-treated by protein preference Wizard in Schrodinger, eliminating the crystal water and adding missing hydrogen/side chain atoms, and assigning appropriate charge and protonation states for acidic and basic amino acid residues at pH 7.0, followed by energy minimization of the enzyme structure using OPLS-2005 force field, and finally prediction of the active site of the protein with Sitemap. Meanwhile, the structural conformation of the forsythin and the derivatives thereof is optimized by using the LigPrep module of Schrodinger.

(2) Molecule docking: virtual screening based on molecular docking was performed using the Glide workflow of Maestro 11.5. Docking calculations for phillyrin and its derivatives (fig. 1) were performed by using the "ultra precision" mode (XP) of Glide.

(3) And (3) docking fraction: the interaction between the small molecule and the target protein is evaluated by a 'Glide-score' score function value, the function comprehensively considers the interactions such as hydrogen bond, hydrophobicity, van der Waals force and the like, and the larger the absolute value of the function is, the more stable the docking complex of the small molecule and the target protein is and the better the matching binding effect is.

(4) And (3) analyzing a target point: and key amino acids of the KD-1 and the derivatives thereof and the key target proteins of the novel coronavirus are analyzed according to the docking result of the KD-1 and the derivatives thereof and the key target proteins of the novel coronavirus so as to provide theoretical basis and important reference for clinical prevention, diagnosis and treatment of the infection of the novel coronavirus.

2 expression and purification of proteins

(1) Cloning: the full-length gene (UniprotKB-P0 DTD1, residues 3264-3569) encoding SARS-CoV-2 CL pro protein has been optimized and synthesized for the E.coli expression system (Wuhan Jin Kairui bioengineering, inc.).

(2) Expressing: the expression plasmid was transformed into E.coli BL21 (DE 3) cells, which were then cultured in LB medium containing 100. Mu.g/ml ampicillin at 37 ℃. When the cells were grown to OD600nm values between 0.6-0.8, 0.5mM IPTG was added to the cell culture to induce expression at 30 ℃ at 180 rpm. After 10 hours, cells were collected by centrifugation at 3,000g.

(3) And (3) purification: the cell pellet was resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 150mM NaCl,2mM BME), lysed by ultrasonication, and then centrifuged at 13,000g for 30 minutes. The supernatant was loaded onto a Ni-NTA affinity column, washed with 20mM imidazole in resuspension buffer, and then His-tagged 3CL pro protein was eluted with 300mM imidazole in lysis buffer (50 mM Tris-HCl pH 7.0, 150mM NaCl). The 3CL pro protein was further purified by ion exchange chromatography. The 3CL pro protein was finally obtained as a mixture of monomers and dimers and stored in solution.

3 spectroscopic experiments

In the experiment, the JASCO-V560 series ultraviolet-visible spectrophotometer is used for researching the structural change of the protein. The uv absorption spectrum of the protein has two absorption peaks, the strong absorption peak at 210nm reflects the framework conformation of the protein due to the n-pi + transition of C = O in the peptide bond. The weak absorption peak at 280nm occurs as a result of the pi-pi transition caused by the absorption of light by the aromatic amino acids (Trp, tyr and Phe).

In the experiment, the buffer solution is used as a reference, and the ultraviolet visible absorption spectrum of the protein solution and the phillyrin protein mixed solution is scanned within the wavelength range of 200-700 nm. To a quartz cuvette was added 2ml Gel Buffer (50 mM Tris-HCl pH 7.0, 150mM NaCl) and 10. Mu.L of 3CL pro protein was added at a concentration of 10 ^-5 M, and measuring the absorbance value, and adding 10 mul of phillyrin and the derivative thereof respectively at the same time with the concentration of 10 ^-4 And M, measuring the change of absorbance.

3. Results of the experiment

The life cycle of COVID-19 is that S protein on the surface of COVID-19 is combined with angiotensin converting enzyme 2 (ACE 2), and the combined protein invades cells to release genetic material RNA through the endocytosis process of the cells, and the genetic material RNA is successfully replicated to generate virus under the participation of 3CL pro protein. This experiment seeks to prevent the 3CL pro protein from participating in the genetic material RNA, and further develops a medicament against COVID-19. The experiment selects the optimal target locus KD-1 and the derivatives (KD-2-GLU and KD-2-SO) thereof through the crystal structure of the 3CL pro protein ₃ H) The compounds are virtually screened to obtain KD-1, KD-2-GLU and KD-2-SO ₃ H compound 3CL pro protein site binding score, thereby demonstrating the targeting of the compound.

Through computational analysis, 5 potential active sites are found in the three-dimensional structure of the 3CLpro protein, wherein site 1 is a site to which a drug molecule is easy to bind. KD-1 is docked to 5 potential active sites of 3CLpro protein, all 5 active sites are successfully docked, and the docking scores are shown in Table 3-1. The Glide Score of KD-1 at the active site 1 of 3CLpro protein is obviously higher than that of other active sites of 3CLpro protein, and the active site 1 of 3CLpro protein is selected as the theoretical value of molecular docking. KD-2-GLU and KD-2-SO ₃ The calculation and analysis of the H compound are the same as above.

TABLE 3-1 KD-1 docking scores with 5 potential active sites of 3CLpro protein

KD-1, KD-2-GLU and KD-2-SO are selected ₃ The binding ability of the small molecule and the 3CLpro protein site is evaluated by 'Glide Score', and the higher the absolute value is, the more stable and matched the binding of the small molecule and the 3CLpro protein site are. KD-1, KD-2-GLU and KD-2-SO ₃ The docking scores of the H compound to the active site of the COVID-19 target protein are shown in Table 3-2. Tables 3-2 show KD-1, KD-2-GLU and KD-2-SO ₃ The H compound has significance to the active site binding theoretical score of the core protein 3CL pro of the COVID-19 participating in RNA replication, wherein the KD-1 and KD-2-GLU binding scores are very high, thereby further indicating that the compounds have different degrees of targeting on the core protein 3CL pro.

TABLE 3-2 KD-1 and its derivatives together with 3CLpro target protein site docking score of COVI D-19

Through the three-dimensional simulation and plan view of the molecular docking of KD-1 and 3CLpro target proteins, it can be seen that KD-1 compound enters the molecular pocket of the protein (FIG. 2. Upper). KD-1 forms pi-pi conjugation with the amino acid His41 residue of the active site of the 3CL pro protein, forms 3 hydrogen bonds with the peptide bonds of Gly143, asn142, glu166, and forms hydrophobic interactions with Met165 to bind to the 3CL pro protein (FIG. 2. Bottom). The different amino acid residues, such as His, gly, asn, glu, met and the like, can be matched with the structure of KD-1 in space, thereby determining the conformation of the complex, and supposing that the KD-1 compound targets the 3CL pro protein and prevents the 3CL pro protein from participating in the COVID-19 in RNA replication to generate viruses.

Through the three-dimensional simulation diagram and the plan view of the molecular butt joint of the KD-2-GLU and the 3CLpro target protein, the KD-2-GLU enters the molecular pocket of the protein (figure 3. Above). The peptide bonds of KD-2-GLU and the active sites Gly143, thr43 and Thr25 of the 3CL pro protein form 3 hydrogen bonds, and form hydrophobic interaction with Tyr54 to be combined with the 3CL pro protein (figure 3. Lower). Indicating that different amino acid residues, such as Gly, thr, and Tyr, can be spatially matched to the structure of KD-2-GLU to determine the conformation of the complex. The KD-2-GLU compound is presumed to target 3CL pro protein and prevent the 3CL pro protein from participating in COVID-19 to participate in RNA replication to generate virus.

By molecular docking of KD-2-SO ₃ Three-dimensional simulation and plan view of the H and 3CLpro target proteins show that KD-2-GLU enters the molecular pocket of the protein (FIG. 4. Top). KD-2-SO ₃ H forms hydrogen bond interaction with Gly143, asn142, and hydrophobic interaction with Tyr54, val42, cys44, cys145 (FIG. 4. Bottom). Indicating that different amino acid residues, e.g., gly, asn, val, etc., may be spatially related to KD-2-SO ₃ H, thereby determining the conformation of the complex.

The interaction of KD-1 and its derivatives with the active site of 3CLpro protein is shown in tables 3-3. The results show that phillyrin and forsythinThe interaction between the seed derivative and the active site of the 3CLpro protein is not completely hydrophobic, but also undergoes hydrogen bonding and pi-pi conjugation, possibly contributing to the stabilization of the complex through hydrogen bonding and pi-pi conjugation. KD-1, KD-2-GLU and KD-2-SO were found ₃ The H compound and the 3CLpro protein mutually interact through a GLY143 hydrogen bond, and the hydrogen atom distance in the molecule is respectively

And

however, hydrophobic and electrostatic interactions are in the 3CLpro protein and KD-1, KD-2-GLU and KD-2-SO ₃ The binding process of H compounds plays an important role, and different compounds have different residues bound with 3CLpro protein, which explains the difference of their binding results.

TABLE 3-3 KD-1 and its derivatives interaction with 3CLpro protein

The protein after disruption was purified by nickel column and the molecular weight was 34kd as shown in FIG. 5 (left). The 3CL pro protein was detected as sample 3CL pro protein by SDS PAGE after ion exchange chromatography at a protein concentration of 65mg/ml in FIG. 5 (right).

Performing KD-1, KD-2-GLU and KD-2-SO in vitro by an ultraviolet-visible absorption test method based on molecular docking virtual sieve results ₃ And H compound and 3CLpro protein binding test further proves that the compound has targeting property on the 3CLpro protein. FIG. 6 shows the measurement of KD-1 and 3C by UV-Vis spectroscopyAbsorbance of Lpro protein and mixtures thereof. The result shows that the 3CL pro protein has an absorption peak at 278nm, and after KD-1 is added, the absorbance is increased, and the peak shifts. The interaction of KD-1 with 3CL pro protein may inhibit the activity of the enzyme. KD-1 and 3CL pro may form a new complex, and KD-1 may hydrogen bond or hydrophobically bind to 3CL pro protein.

FIG. 7 shows the absorbance of KD2-GLU and 3CLpro and mixtures thereof as determined by UV-Vis spectroscopy. The results show that 3CL pro protein has an absorption peak at 278nm, and the absorbance increases and the peak shifts after KD2-GLU is added. KD2-GLU interacts with 3CL pro protein, possibly inhibiting the activity of the enzyme. KD2-GLU and 3CL pro may form a new complex, and both may undergo hydrogen bonding and hydrophobic binding.

FIG. 8 shows the measurement of KD2-SO by UV-Vis spectroscopy ₃ Absorbance of H and 3CLpro and mixtures thereof. The results show that KD2-SO is added ₃ After H, the absorbance increased and the peak of 3CL pro protein shifted. KD2-SO ₃ The interaction of H with the 3CL pro protein may inhibit the activity of the enzyme. KD2-SO ₃ H may hydrogen bond or hydrophobically bind to 3CL pro protein, and a new complex may be formed between the two.

4. Conclusion of the experiment

The results of molecular docking show that KD-1, KD-2-GLU and KD-2-SO ₃ The H compound has significance to the binding theory score of the core protein 3CL pro protein of the COVID-19 virus involved in RNA replication, wherein the binding scores of KD-1 and KD-2-GLU are high. The analysis shows that KD-1, KD-2-GLU and KD-2-SO ₃ The H compound and the peptide bond in the active site of the 3CL pro protein are subjected to hydrogen bond and hydrophobic combination, and form pi-pi conjugation with amino acid residues, and theoretical data of the H compound can prove that the 3CL pro protein structure is changed. In vitro spectroscopic experiments show that the results of KD-1, KD-2-GLU and KD-2-SO ₃ The H compound has shift and increase phenomena on the characteristic peak of 3CL pro protein at 278nm, and the KD-1 compound is the most obvious. Thus KD-1, KD-2-GLU and KD-2-SO ₃ The H compounds all haveThe compound which has a targeting effect on the 3CL pro protein and prevents the 3CL pro protein of the COVID-19 virus from participating in the replication of the virus can be a targeting inhibitor of the COVID-19 virus.

Example 4 in vivo efficacy Studies against New coronavirus

1. Experimental Material

Mice: hACE2 mice, 6-7 weeks old, 20-40g, 120 males total, experimental animal supplier: jiangsu Jiejiaokang biotechnology limited, laboratory animal production license: SCXK (su) 2018-0008, laboratory animal certification No.: no.320727201100243581, feed supplier: jiang Sumei disen biopharmaceutical, inc.

Medicine preparation: a forsythin/forsythiaside composition, wherein the weight ratio of forsythin to forsythiaside is 90.

2. Experimental methods

(1) Protective effect of medicament on disease progression of COVID-19 mice

hACE2 transgenic C57BL/6 mice were divided into normal group, SARS-CoV-2 infected group, drug 80mg/kg, 40mg/kg dose group, positive control group (Reidesvir 50 mg/kg), 8 mice per group. In addition to the normal group given PBS by nasal drip, other groups of mice were infected by nasal drip 10 ⁵ PFU SARS-CoV-2 virus. The drug group mice were gavaged with phillyrin 2 hours after infection for 5 consecutive days, 1 time/day. Body weight changes were recorded daily after infection and 5-day mortality was calculated.

(2) Drug effect research on excessive inflammation induced by COVID-19 mouse infection

ACE2 transgenic C57BL/6 mice were divided into normal group, SARS-CoV-2 infected group, drug 80mg/kg, 40mg/kg dose group, and positive control group (Reidesvir 50 mg/kg). In addition to the normal group given PBS by nasal drip, the other groups of mice infected with nasal drip 10 ⁵ PFU SARS-CoV-2 virus. The drug group mice were gavaged with phillyrin 2 hours after infection for 5 consecutive days, 1 time/day. Dissecting the animal on the 5 th day after infection, taking the lung, homogenizing the lung tissue and detecting the virus titer; extracting total RNA from lung tissue homogenate supernatant by Trizol method, and detecting related inflammatory factor mRNA by RT-qPCRExpression of (2).

3. Results of the experiment

(1) The results of the anti-neocoronavirus death protection study of the drug

The results of the death protection are shown in Table 4-1 and show that: the protective effect of the drug on mice infected with the new coronavirus is 87.5 percent and 42.86 percent respectively by two dosage groups (80 mg/kg and 40 mg/kg); 71.43% of mice in a virus infection group die, and the death protection rate of a drug 80mg/kg group is equivalent to that of the Reineckevir.

Table 4-1 test results for death protection in mice infected with the novel coronavirus (n = 3)

(2) Detection result of drug on lung tissue virus titer of mice infected by new coronavirus

As shown in the table 4-2, the pulmonary virus titer of the infected mice is remarkably reduced by the administration groups of the two drugs (80 mg/kg and 40 mg/kg), and the pulmonary virus titer inhibition of the administration group of 80mg/kg is not statistically different from that of the Reidesvir.

TABLE 4-2 measurement results of the titer of the novel coronavirus-infected mouse lung tissue virus: (

n＝3)

Note: p <0.05, P <0.01, P <0.001, compared to the virus-infected group

(3) Detection result of drug on excessive inflammation induced by novel coronavirus infected mouse

The detection results of the inflammation indexes of the infected mice are shown in the table 4-3, the expression of IL-1 beta, IFN-gamma, MCP-1 and IFN-alpha of a virus infection group is obviously increased on the fifth day of the mice infected by the new coronavirus, and after the medicine is dried, two administration dose groups (80 mg/kg and 40 mg/kg) have the inhibition effect on IL-1 beta, IFN-gamma, MCP-1 and IFN-alpha inflammation factors; and the capability of inhibiting the over-expression of inflammatory mediators in an 80mg/kg administration group is better than that of the Redexilvir.

TABLE 4-3 Effect of drugs on the expression levels of IL-1 β, IFN- γ, MCP-1 and IFN- α mRNA in homogenates of lungs from infected mice: (

n＝3)

Note: p <0.05, P <0.01, P <0.001, compared to the virus-infected group

4. Conclusion

The experimental results show that the medicine has a remarkable protection effect on the death of mice infected by the new coronavirus, the death protection rate of the mice infected by the new coronavirus reaches 87.5 percent, and the medicine is equivalent to Reidesxi Wei Liaoxiao, can remarkably inhibit the titer of the lung tissue virus of the mice infected by the new coronavirus, and can perform the mRNA expression of excessive inflammatory factors IL-1 beta, IFN-alpha, MCP-1 and IFN-gamma induced by the infection of the new coronavirus, thereby playing a role in treating the infection of the new coronavirus.

Claims

Use of phillyrin and/or a derivative thereof in the manufacture of a reagent for inhibiting the 3CLpro protein of the COVID-19 virus.
The use of claim 1, wherein the derivative is KD-2-GLU or KD-2-SO ₃ H。
A method of inhibiting 3CLpro protein of covi-19 virus comprising the step of contacting 3CLpro protein of covi-19 virus with phillyrin and/or a derivative thereof.
In the application ofThe method of claim 3, wherein the derivative is KD-2-GLU or KD-2-SO ₃ H。
Application of phillyrin/phillygenin composition in preparing medicine for resisting COVID-19 virus or treating diseases caused by COVID-19 virus is provided.
The use as claimed in claim 5, wherein the weight ratio of forsythin to forsythiaside in the forsythin/forsythiaside composition is 2-98: 2 to 98.
The use according to claim 5 of a phillyrin/phillygenin composition as the sole pharmaceutically active ingredient.
The use according to claim 5, wherein the medicament is in the form of a tablet, capsule, pill, powder, granule, syrup, solution, emulsion, injection, spray, aerosol, gel, cream, cataplasma, rubber patch or patch.
The use of claim 5 wherein the phillyrin/phillygenin composition inhibits the 3CLpro protein of the COVID-19 virus.
A method for resisting COVID-19 virus or for treating a disease caused by the COVID-19 virus, comprising administering to a patient in need thereof an effective amount of a phillyrin/phillygenin composition.
A method for resisting COVID-19 virus or for treating a disease caused by the COVID-19 virus, comprising administering to a patient in need thereof an effective amount of a medicament comprising a forsythin/forsythiaside composition.
The method of claim 10 or 11, wherein the weight ratio of phillyrin to phillygenin in the phillyrin/phillygenin composition is 2-98: 2 to 98.
A method according to claim 10 or 11 wherein the forsythin/forsythiaside composition is used as the sole pharmaceutically active ingredient.
The method of claim 10 or 11, wherein the medicament is in the form of a tablet, capsule, pill, powder, granule, syrup, solution, emulsion, injection, spray, aerosol, gel, cream, cataplasm, rubber patch or patch.
The method of claim 10 or 11 wherein the phillyrin/phillygenin composition inhibits the 3CLpro protein of COVID-19 virus.
Phillyrin/phillygenin compositions against the COVID-19 virus or for treating diseases caused by the COVID-19 virus or inhibiting the 3CLpro protein of the COVID-19 virus.
The composition of claim 16, wherein the weight ratio of phillyrin to phillygenin in the phillyrin/phillygenin composition is 2-98: 2 to 98.
A medicament comprising a phillyrin/phillygenin composition for use against the COVID-19 virus or for the treatment of diseases caused by the COVID-19 virus or for inhibiting the 3CLpro protein of the COVID-19 virus.
The medicament of claim 18, wherein the weight ratio of phillyrin to phillygenin in the phillyrin/phillygenin composition is 2-98: 2 to 98.
A medicament as claimed in claim 18, wherein the phillyrin/phillygenin composition is provided as the sole pharmaceutically active ingredient.
The medicament of claim 18, wherein the medicament is in the form of a tablet, capsule, pill, powder, granule, syrup, solution, emulsion, injection, spray, aerosol, gel, cream, cataplasm, rubber patch or patch.